1. Field of the Invention
The present invention relates to a major
Brugia malayi enzyme which is a biologically active, recombinant asparaginyl-tRNA synthetase. This synthetase functions specifically to aminoacylate tRNA with the amino acid asparagine. Isolation of the filarial enzyme facilitates exploration of structure-function relationships relevant to cognate tRNA binding and biological activity for new drug development against filarial diseases. Further, the synthetase is used in an assay for inhibition or promotion of activity of the enzyme by various compounds. The enzyme is used for producing antibodies used in assays to detect filarial nematodes in biological tissues. The synthetase can also be used to detect antibodies to the synthetase in a biological tissue.
2. Description of Related Art
Lymphatic filariasis is caused by infection with the filarial nematodes Brugia malayi, Brugia timori, Wuchereria bancrofti and Onchocerca volvulus which collectively infect about 200 million persons throughout the world. The filarial nematodes cause lymphatic filariasis (elephantiasis) or onchocerciasis (river blindness). New control strategies for these parasites seek to characterize important parasite antigens and biochemical pathways to serve as targets for vaccine or new drug development (Philipp, M., et al., Ann Rev. Microbiol. 42 685-716 (1988)). There is also a need to develop assays for filarial nematodes. The aminoacyl-tRNA synthetases (AARS) are a group of enzymes whose primary function is to specifically attach amino acids to the 3'-adenosine of their cognate tRNAs in a two step process which involves the formation of an enzyme bound aminoacyl-adenylate intermediate (Carter, T. W., Ann. Rev. Biochem. 62 715-748 (1993)). These enzymes thus play a crucial role in ensuring the fidelity of protein biosynthesis by discriminating against incorrect aminoacylation and thus are fundamental to the growth and replication of all forms of life. The structural basis for AARS specificity lies primarily in mechanisms responsible for the recognition of cognate tRNAs. The 20 AARS of both prokaryotes and eukaryotes can now be divided into two classes based on short peptide sequence motifs and catalytic domain topologies: a Rossmann fold for class I and an antiparallel .beta. fold for class II (Eriani, G., et al., Nature 347 203-206 (1990); and Cusack, S., et al., Nature 347 249-255 (1990)).
In the course of immunological studies of filarial parasite antigens, the gene encoding an immunodominant 63 kDa peptide in B. malayi was also found within the genome of another major human filarial parasite, O. volvulus the organism which causes onchocerciasis (Nilson, T. W., et al., Proc. Natl. Acad. Sci. USA 85 3604-3607 (1988); and Kron, M., et al., Mol. Biochem. Parasit. 52 289-292 (1992)). When originally described, the derived amino acid sequence of the B. malayi gene product was thought to have no similarity with previously known proteins. Subsequent to recognition of a second class of AARS in 1990 (Eriani, G., et al., Nature 347 203-206 (1990); and Cusack, S., et al., Nature 347 249-255 (1990)), reanalysis of the B. malayi antigen primary structure revealed the presence of one, and possibly two, allowing for the possibility of a frame-shift error in the determination of the gene sequence, structural motifs characteristic of the class II aminoacyl-tRNA synthetases which strongly suggested that the antigen was an asparaginyl-tRNA synthetase (Cusack, S., et al., Nucleic Acids Research 19 3489-3497 1991)). Previously, only a truncated 55 kDa portion of the B. malayi peptide had been expressed for immunological studies. There is a need for a pure form of the complete asparaginyl-tRNA synthetase for the reasons discussed above.